Nfix Is Required for Hematopoietic Stem- and Progenitor Cell in Vivo Repopulating Potential.

Abstract 2320

Hematopoietic stem cells (HSCs) are both necessary and sufficient to sustain the complete blood system of vertebrates. Specified at several locations during fetal development, they ultimately congregate in the fetal liver for rapid expansion. Around birth HSCs relocate to the bone marrow (BM) and enter a state of cellular quiescence, cycling intermittently to supply progenitor cells, which differentiate into the distinct blood lineages. Due to their regenerative potential, HSCs are heavily utilized in the clinic for bone marrow transplants (BMTs) to treat a variety of diseases. However, a lack of suitable donors and inefficiency in recipient engraftment currently limit this life saving therapy. To improve BMT regimens, a better understanding of regulators of HSC BM engraftment is required.

Recently, we examined the gene expression patterns of HSCs as they emerge throughout murine ontogeny (McKinney-Freeman et al., Cell Stem Cell, in press). We observed that the transcription factor Nfix, a member of the nuclear factor I (NFI) family of transcription factors never before linked to HSC biology, was highly expressed by both fetal liver and BM HSCs. These data suggest that Nfix may play a novel role in regulating HSC function in the BM and/or fetal liver. To test this hypothesis, HSCs were enriched from E14.5 fetal liver or adult BM (Lineage-, c-kit+, Sca-1+ (LSK) cells) and then transduced with lentiviruses carrying shRNAs targeting Nfix. Twenty-four hours post-transduction, cells were injected into lethally irradiated mice along with untransduced BM LSK competitor cells congenic at the CD45 allele. The peripheral blood of recipient mice was then analyzed periodically over 16 weeks for engraftment of the Nfix-depleted cells. Depletion of Nfix by two independent shRNA (confirmed by Western blot analysis to deplete NFIX protein levels to <20% of baseline) resulted in a significant decrease in the repopulating activity of BM LSK cells relative to LSK cells transduced with either of two independent control shRNAs. As early as two weeks post-transplant, a 22% +/− 5% (p=0.03) reduction in repopulating activity was observed. By 16 weeks post-transplant, this reduction in repopulating potential had gradually increased to 55% +/−8% (p<0.0001) in four independent experiments. Depletion of Nfix in fetal liver-derived LSK cells resulted in a similar loss of repopulating potential. Critically, in vitro analysis of BM LSK expansion in liquid culture and differentiative potential, as analyzed by the methylcellulose based colony forming assay, revealed no differences in the activity of LSK cells transduced with Nfix-specific shRNAs compared to controls. Thus, it is unlikely that the observed decrease in BM repopulating activity is due to general cytotoxicity resulting from Nfix depletion or a block in differentiation. Concordantly, lineage analysis of peripheral blood of recipients showed no significant differences in the percentage of the major blood lineages derived from LSK cells transduced with Nfix-specific shRNAs compared to controls. Thus, the observed decrease in repopulating activity likely occurs at the level of HSCs and multipotent progenitors. In agreement with this conclusion, when BM of recipients transplanted with Nfix-depleted LSK cells are examined 4 and 16 weeks post transplant, a loss of phenotypic HSCs (LSK/CD150+/CD48-) relative to controls is evident.

The loss of repopulating potential by Nfix-depleted cells as early as two weeks post-transplant suggests that Nfix may be involved in either the homing/lodgment of HSCs in the BM or their ability to expand soon after incorporation into the stem cell niche. We are presently teasing out the molecular mechanism behind this phenomenon. Furthermore, to examine a role for Nfix during HSC homeostasis, we are currently analyzing mice in which Nfix has been conditionally ablated in the hematopoietic compartment. Finally, functional analysis of two other members of the NFI gene family shown by our array analysis to be expressed by BM HSC, Nfia and Nfic, will further assess a role for this gene family in the regulation of HSCs.

In summary, we have for the first time established a role for a member of the NFI gene family, Nfix, in HSC biology, as evident by a decrease in BM repopulating activity in Nfix-depleted HSCs. By dissecting the precise role of Nfix in HSC biology, we will glean insights that could improve our understanding of graft failure in clinical BMTs.